Multifunctionality of Hemocyanin in Mollusca and Arthropoda: Structural Evolution, Phenoloxidase Activity, and Therapeutic Bioactivities

Abstract Multifunctional proteins that support both immunological and metabolic processes are essential for invertebrate life. Hemocyanins and phenoloxidases, both of which belong to the type-3 copper protein family, serve as the best examples of this duality. Hemocyanins, prominent in molluscs and arthropods, are colossal multimeric glycoproteins that reversibly bind oxygen, imparting a characteristic blue colour to the hemolymph. Structural studies have revealed their highly ordered architecture, formed from conserved subunits arranged into decameric and higher-order assemblies. Notably, hemocyanins have undergone extensive gene duplication and domain specialization, leading to both respiratory and non-respiratory roles. Beyond their respiratory role, hemocyanins exhibit remarkable functional versatility and have been associated with diverse biological activities, including antimicrobial, antiviral, and anticancer properties, highlighting their potential biomedical significance. Parallel to this, phenoloxidases mediate melanization, a critical defence mechanism in invertebrates. The prophenoloxidase activity system not only triggers melanin formation upon pathogen invasion but also generates bioactive fragments with cytokine-like and antimicrobial properties. Intriguingly, hemocyanins can exhibit phenoloxidase-like activity under specific conditions, blurring the functional lines between these proteins. Phylogenetic analyses suggest that hemocyanins in both Mollusca and Arthropoda originated from ancestral phenoloxidase-like enzymes belonging to the type-3 copper protein family. During evolution, these proteins underwent lineage-specific diversification, giving rise to distinct structural and functional adaptations in molluscan and arthropod hemocyanins. In arthropods, additional evolutionary modifications produced related proteins such as hexamerin storage proteins, highlighting the remarkable evolutionary plasticity of this protein family. This review emphasizes the structural intricacy, evolutionary trajectories, and immune significance of hemocyanins and phenoloxidases, showcasing how a single protein framework has diversified to satisfy the physiological and immunological needs of invertebrate life.